Mutant phosphoribosylpyrophosphate synthetase and method for producing L-histidine

Inactive Publication Date: 2005-08-11
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025] It is a further object of the present invention to provide the method as described a

Problems solved by technology

However, there are no reports describing mutant bacterial PRPP synthetase which is feedback-resistant to purine nucleotid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of the Wild-Type prsA Gene from E. coli and Construction of the Mutant prsDA and prsDS Genes

[0066] The entire nucleotide sequence of E. coli strain K-12 has been reported (Science, 277, 1453-1474, 1997). Based on the reported nucleotide sequence, the primers depicted in SEQ ID No. 3 (primer 1) and SEQ ID No.4 (primer 2) were synthesized and used for amplification of prsA gene. The primer 1 contains a BglII recognition site introduced at the 5′ thereof. The primer 2 contains a XbaI recognition site introduced at the 5′-end thereof.

[0067] Chromosomal DNA of E. coli K12 was used as template for PCR, and was prepared by an ordinary method. PCR was carried out on the Applied Biosystems GeneAmp PCR System 2400 under the following conditions: initial DNA denaturation at 95° C. for 3 min; followed by 30 cycles of denaturation at 95° C. for 30 sec, annealing at 60° C. for 60 sec and elongation at 72° C. for 120 sec; and the final polymerization for 7 min at 72° C. using Taq polymer...

example 2

Effect of Enhanced Expression of the purH Gene on Histidine Production

[0070] Three L-histidine-producing plasmid-less strains containing additional copies of the prsA, prsDA or prsDS genes integrated into the bacterial chromosome were constructed. The L-histidine producing E. coli strain 80 was used as a parental strain for integration of the prsA, prsDA and prsDS genes into the bacterial chromosome. The strain 80 is described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 113545 Moscow, 1st Dorozhny proezd, 1) under accession number VRPM B-7270.

[0071] Integration of the genes into the chromosome of strain 80 was performed in two steps. For the first step, the histidine-producing strain 80 was transformed with a helper plasmid containing replicon rep(p15A), transposase gene (genes cts62, ner, A, B from phage Mu-cts) and containing TetR marker. For the second step, the resulting strain was transformed with plasmid pM...

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PUM

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Abstract

The present invention relates to a mutant bacterial PRPP synthetase which is resistant to feedback by purine nucleotides, and a method for producing L-histidine using the bacterium of the Enterobacteriaceae family wherein the L-amino acid productivity of said bacterium is enhanced by use of the PRPP synthetase which is resistant to feedback by purine nucleotides, coded by the mutant prsA gene.

Description

[0001] This application claims the benefit of provisional application 60 / 587,492, filed Jul. 14, 2004.BACKGROUND OF THE INVENTION [0002] 1. Technical Field [0003] The present invention relates to a method for producing L-amino acid, such as L-histidine. More specifically, the present invention relates to a novel feedback-resistant enzyme involved in the biosynthesis of purines and L-histidine. More specifically, the present invention concerns a new feedback-resistant mutant phosphoribosylpyrophosphate synthetase (PRPP synthetase) from E. coli. The invention also relates to a method for producing L-histidine by fermentation using bacterial strains containing the novel feedback-resistant enzyme. [0004] 2. Background Art [0005] Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources or mutants thereof, which are modified to enhance production yields of L-amino acids. [0006] Many techniques to enha...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12P13/24
CPCC12P13/24C12N9/1235
Inventor KLYACHKO, ELENASHAKULOV, RUSTEMKOZLOV, YURI
Owner AJINOMOTO CO INC
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